With the advances in nanoparticle (NP) development for use in healthcare, in vitro and in vivo systems to test their toxicity and bioactivities are needed. The goal of the proposed research is to develop an avian test-system to monitor NP toxicity and bioactivities in vivo. The most unique feature of this avian test-system is repeat access to a complex dermal tissue, namely the pulp of growing feathers (GF). Both injection and collection of GF are considered minimally invasive procedures. Events taking place in injected feathers can be monitored by injecting multiple GF at one time and then collecting GF at various times post-injection for ex vivo analysis. Moreover, blood sampling along with collection of injected GF allows for simultaneous qualitative and quantitative assessment of events taking place in GF and in the blood circulation. Hence, this avian test- system constitutes a unique opportunity to assess and monitor in vivo NP toxicity and bioactivities in the same individual using minimally invasive, non-terminal procedures. In chickens, the ensheathed living portion of a GF is a define unit of tissue approx. 8-10 mm in height with a 2-3 mm diameter. The dermis-like pulp is bordered by an epidermal layer, contains the vascular supply of the GF and is an immunologically active site capable of recruitment of leukocytes from the circulation. Moreover, GF can easily be removed by pulling it from its follicle, a procedure that is less invasive than taking a blood sample, and the feather subsequently regenerates. Hence, we hypothesized that GF can serve as an in vivo test-tube suitable for minimally invasive examination of biological activities of NP in vivo. The broad objective of the proposed research is to demonstrate the unique opportunities of the avian system to monitor and study NP bioactivities and interactions in vivo and to establish the chicken as a suitable and convenient intermediate animal model for biomedical application of NP. Specifically, in Aim 1, toxicity, activities and interactions of NP ad antigen-conjugated NP (Ag-NP) with avian cellular and soluble immune system components will be determined in vitro. In Aim 2, the physiological, cellular and molecular events (e.g. inflammatory processes, complement activation, cytokines production, vascular changes) initiated by injection of NP and Ag-NP into GF will be assessed and monitored in GF and blood. In Aim 3, qualitative and quantitative aspects of primary and memory cell-mediated (profiles of effector cells and activities) and humoral (type and levels of antibodies) immune responses to intramuscularly injected Ag-NP will be assessed in GF and blood. In Aim 4, the possibility of using GF to monitor NP-targeted delivery will be explored by intravenous injection of NP-conjugated with Ag-specific antibody (Ab-NP) and injection of target Ag into GF. The proposed project will establish the chicken as a suitable and convenient intermediate animal model to monitor NP activities and effects in vivo using minimally invasive procedures. In addition, the proposed research will provide new knowledge on NP toxicity, NP molecular cellular interface interactions, NP immunomodulatory effects on innate and adaptive immune responses, and on NP targeting and delivery.